The human cytidine deaminases APOBEC3G and 3F (A3G/F) are antiviral proteins that are expressed in human tissues and inhibit HIV replication by enzymatic and nonenzymatic mechanisms. Acting enzymatically, A3G/F edits HIV DNA, causing catastrophic hypermutation. Large clinical studies reveal that high levels of A3G/F-mediated hypermutation correlate with lower viral loads and higher CD4 counts, suggesting that this type of hypermutation is associated with reduced disease progression. However, because few studies have focused on A3G expressed endogenously in primary cells, little is known about which pools of A3G/F in primary cells are enzymatically active, how A3G/F activity is regulated in primary cells, and how physiologically relevant events like T cell activation affect A3G/F enzymatic activity. Using a high-throughput assay for measuring A3G enzymatic activity, we demonstrated that deaminase activity of endogenously expressed A3G in resting and fully activated primary human T cells is inhibited, suggesting that A3G is negatively regulated in primary T cells. Our preliminary data demonstrate that in human peripheral blood mononuclear cells (PBMCs), A3G is post-translationally activated at short times after T cell receptor stimulation. In this application, we propose a systematic study of A3G/F in human PBMCs and T cells. Specifically, we will 1) determine which signaling pathways activate and inhibit A3G enzymatic activity in T cells;2) examine whether A3G/F in PBMCs that are the target of infection contributes to hypermutation of HIV DNA;3) determine which A3 proteins are packaged into virus produced by primary human T cells and macrophages using specific antisera we have generated;and 4) quantify the enzymatic and nonenzymatic effects of A3G/F in virions produced by PBMCs by systematically measuring enzymatic activity and infectivity of these virions, as well as HIV hypermutation and reverse transcripts in target cells infected by these virions. The proposed studies should greatly contribute to our knowledge of how A3G/F functions in primary human cells and will add significantly to our incomplete understanding of A3G/F in infected patients. PUBLIC HEALTH RELEVANCE: The human cytidine deaminases APOBEC3G and 3F (A3G/F) are antiviral proteins that inhibit viral replication in part by causing catastrophic hypermutation of HIV DNA through DNA editing. Because high levels of A3G/F-mediated hypermutation of HIV DNA in infected patients correlate with lower viral loads and higher CD4 count, it will be important to understand how hypermutation arises in vivo;however, A3G/F in primary human cells is relatively understudied. The proposed studies will address how A3G/F functions in primary human peripheral blood mononuclear cells (PBMCs) by determining which pools of A3G/F are enzymatically active in HIV infected PBMCs and the virions they produce, and whether signaling pathways regulate A3G/F enzymatic activity and hypermutation of HIV DNA in infected PBMCs.